Design and purification of active truncated phosphoinositide 3-kinase gamma protein constructs for structural studies.

Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase that plays a crucial role in cell migration, chemotaxis, oxidative burst and myocardial contractility. It is activated downstream of G protein-coupled receptors (GPCRs) and small GTPases of Ras superfamily. PI3Kγ is a heterodimer composed of a catalytic and a regulatory subunit that is expressed mostly in hematopoietic cells and in the heart. Although it has attracted a lot of attention because of its link with tumor inflammation and heart diseases, its regulation is still not fully understood. This can be attributed to the absence of high-resolution structural details of the PI3Kγ heterodimer. Here we describe the design and purification of PI3Kγ constructs where flexible loops in the regulatory subunit have been removed based on structural information obtained by hydrogen/deuterium exchange - mass spectrometry (HDX-MS). The soluble constructs retain both basal activity and sensitivity to GPCR stimulation, and are thus an optimal tool to further explore their regulation using a structure-based approach.

Using Hydrogen-Deuterium Exchange Mass Spectrometry to Examine Protein-Membrane Interactions.

Many fundamental cellular processes are controlled via assembly of a network of proteins at membrane surfaces. The proper recruitment of proteins to membranes can be controlled by a wide variety of mechanisms, including protein lipidation, protein-protein interactions, posttranslational modifications, and binding to specific lipid species present in membranes. There are, however, only a limited number of analytical techniques that can study the assembly of protein-membrane complexes at the molecular level. A relatively new addition to the set of techniques available to study these protein-membrane systems is the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS experiments measure protein conformational dynamics in their native state, based on the rate of exchange of amide hydrogens with solvent. This review discusses the use of HDX-MS as a tool to identify the interfaces of proteins with membranes and membrane-associated proteins, as well as define conformational changes elicited by membrane recruitment. Specific examples will focus on the use of HDX-MS to examine how large macromolecular protein complexes are recruited and activated on membranes, and how both posttranslational modifications and cancer-linked oncogenic mutations affect these processes.